Procaspase 3 activation by combination therapy

ABSTRACT

The invention provides compositions and methods for the induction of cell death, for example, cancer cell death. Combinations of compounds and related methods of use are disclosed, including the use of compounds in therapy for the treatment of cancer and selective induction of apoptosis in cells. The disclosed drug combinations can have lower neurotoxicity effects than other compounds and combinations of compounds.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/220,361, filed Jul. 26, 2016, issued as U.S. Pat. No. 10,085,977 onOct. 2, 2018, which is a continuation of U.S. patent application Ser.No. 14/383,441, filed Sep. 5, 2014, issued as U.S. Pat. No. 9,399,035 onJul. 26, 2016, which is a National Stage filing under 35 U.S.C. § 371 ofPCT/US2013/029405, filed Mar. 6, 2013, which application claims priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.61/607,098, filed Mar. 6, 2012, which applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, plays a central role in thedevelopment and homeostasis of all multicellular organisms. A frequenthallmark of cancer is resistance to natural apoptotic signals. Dependingon the cancer type, this resistance is typically due to up- ordown-regulation of key proteins in the apoptotic cascade or to mutationsin genes encoding these proteins. Such changes occur in both theintrinsic apoptotic pathway, which funnels through the mitochondria andcaspase-9, and the extrinsic apoptotic pathway, which involves theaction of death receptors and caspase-8. For example, alterations inproper levels of proteins such as p53, Bim, Bax, Apaf-1, FLIP and manyothers have been observed in cancers. The alterations can lead to adefective apoptotic cascade, one in which the upstream pro-apoptoticsignal is not adequately transmitted to activate the executionercaspases, caspase-3 and caspase-7.

As most apoptotic pathways ultimately involve the activation ofprocaspase-3, upstream genetic abnormalities are effectively “breaks” inthe apoptotic circuitry, and as a result such cells proliferateatypically. Given the central role of apoptosis in cancer, efforts havebeen made to develop therapeutics that target specific proteins in theapoptotic cascade. For instance, peptidic or small molecule binders tocascade members such as p53 and proteins in the Bcl family or to theinhibitor of apoptosis (IAP) family of proteins have pro-apoptoticactivity, as do compounds that promote the oligomerization of Apaf-1.However, because such compounds target early (or intermediate to high)positions on the apoptotic cascade, cancers with mutations affectingproteins downstream of those members can still be resistant to thepossible beneficial effects of those compounds.

It would be advantageous for therapeutic purposes to identify smallmolecules that directly activate a proapoptotic protein far downstreamin the apoptotic cascade. This approach could involve a relatively lowposition in the cascade, thus enabling the killing of even those cellsthat have mutations that affect upstream apoptotic machinery. Moreover,such therapeutic strategies would have a higher likelihood of success ifthat proapoptotic protein were upregulated or present at increasedlevels in cancer cells. Thus, the identity of small molecules thattarget the downstream effector protein of apoptosis, procaspase-3, wouldsignificantly aid current cancer therapy.

The conversion or activation of procaspase-3 to caspase-3 results in thegeneration of the active “executioner” caspase form that subsequentlycatalyzes the hydrolysis of a multitude of protein substrates. Activecaspase-3 is a homodimer of heterodimers and is produced by proteolysisof procaspase-3. In vivo, this proteolytic activation typically occursthrough the action of caspase-8 or caspase-9. To ensure that the zymogen(proenzyme) is not prematurely activated, procaspase-3 has a 12 aminoacid “safety catch” that blocks access to the ETD site (amino acidsequence, ile-glu-thr-asp) of proteolysis. This safety catch enablesprocaspase-3 to resist autocatalytic activation and proteolysis bycaspase-9. Mutagenic studies indicate that three consecutive asparticacid residues appear to be the critical components of the safety catch.The position of the safety catch is sensitive to pH, thus upon cellularacidification (as occurs during apoptosis) the safety catch is thoughtto allow access to the site of proteolysis, and active caspase-3 can beproduced either by the action of caspase-9 or through an autoactivationmechanism.

In certain cancers, the levels of procaspase-3 are elevated relative tonormal tissue. A study of primary isolates from 20 colon cancer patientsrevealed that on average, procaspase-3 was upregulated six-fold in suchisolates relative to adjacent non-cancerous tissue. In addition,procaspase-3 is upregulated in certain neuroblastomas, lymphomas, andliver cancers. Furthermore, a systematic evaluation was performed ofprocaspase-3 levels in the 60 cell-line panel used for cancer screeningby the National Cancer Institute (NCI) Developmental TherapeuticsProgram, which revealed that certain lung, melanoma, renal, and breastcancers show greatly enhanced levels of procaspase-3 expression.

Due to the role of active caspase-3 in achieving apoptosis, therelatively high levels of procaspase-3 in certain cancerous cell types,and the intriguing safety catch-mediated suppression of itsautoactivation, small molecules that directly modify procaspase-3 couldhave great applicability in targeted cancer therapy.

Combination therapy has become standard for treatment of cancerpatients. The goal of combination therapy drug cocktail regimes is toachieve a synergistic or additive effect between chemotherapeutics,thereby facilitating shortened treatment times, decreased toxicity, andincreased patient survival. Drugs that act on a single biochemicalpathway are particularly strong candidates for synergy or potentiationas they may mimic “synthetic lethal” genetic combinations. For example,inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1), an enzyme thatfacilitates DNA damage repair, potently synergize with DNA damagingagents as demonstrated in cell culture, animal models, and humanclinical trials. However, there is still a need for more effectivetherapies for the treatment of many forms of cancer, and new synergisticcombinations of anticancer drugs would aid this pursuit. Accordingly,there exists a need to identify new cytotoxic agents that are effectivein killing cancer cells yet protect normal host tissues from theundesired toxicity of the cytotoxic agent.

SUMMARY

The invention broadly provides compounds, compositions, and methods oftherapeutic treatment. In various embodiments, the inventions areapplicable to a variety of cancer diseases and cancer cell types such asbreast, lymphoma, adrenal, renal, melanoma, leukemia, neuroblastoma,lung, brain, and others known in the art. Herein is disclosed, interalia, compositions and methods including small molecules capable ofinducing cell death. In some embodiments, the compositions and methodsinvolve compounds that can interact directly or indirectly withprogrammed cell death pathway members such as procaspase-3. In certainembodiments, the compositions and methods have reduced neurotoxicitycompared to other compounds that interact directly or indirectly withprogrammed cell death pathway members such as procaspase-3.

Combination anticancer therapy can consist of drugs that targetdifferent biochemical pathways, or those that hit different targets inthe same pathway, mimicking “synthetic lethal” genetic combinations. Thecombination of the procaspase-3 activator PAC-1 and a second activeagent has shown considerable synergy toward inducing apoptotic death ofcancer cells, often to a degree well exceeding the additive effect. Thecombination of PAC-1 and a second active agent can be used toeffectively reduce tumor burden in tumor models in which the compoundsalone have minimal or no effect. The data described herein indicate theefficacy of a PAC-1/second agent combination for the treatment of cancerand, more broadly, show that the combinations can be synergistic andprovide significantly heightened therapeutic benefits.

Accordingly, the invention provides a composition comprising:

-   -   (a) the compound PAC-1:

-   -   (b) a second active agent; and (c) a pharmaceutically acceptable        diluent, excipient, or carrier. The second active agent can be,        for example, etoposide, bortezomib, staurosporine, doxorubicin,        tamoxifen, cisplatin, carboplatin, paclitaxel, or another        chemotherapeutic or otherwise active agent recited herein. The        carrier can include water and optional components for        advantageously delivering the actives such as a buffer, a sugar,        solubilization agents such as a cyclodextrin, or various        combinations thereof. In one embodiment, the cyclodextrin is        2-hydroxypropyl-β-cyclodextrin.

The concentration of PAC-1 can be about 0.2 μM to about 5 mM, or about 2μM to about 50 μM, typically about 2.5 μM, about 5 μM, about 7.5 μM,about 10 μM, about 12.5 μM, about 15 μM, about 20 μM, about 25 μM, about30 μM, about 40 μM, or about 50 μM, or a range between any of theaforementioned values. The concentration of the second active agent canbe about 1 nM to about 1 mM, or about 25 nM to about 1 mM, typicallyabout 1 nM, about 2 nM, about 3 nM, about 5 nM, about 10 nM, about 25nM, about 50 nM, about 100 nM, about 250 nM, about 500 nM, about 750 nM,about 900 nM, about 1 μM, about 2.5 μM, about 5 μM, about 7.5 μM, about10 μM, about 12.5 μM, about 15 μM, about 20 μM, about 25 μM, about 30μM, about 40 μM, about 50 μM, about 75 μM, about 100 μM, about 125 μM,about 150 μM, about 200 μM, about 250 μM, about 300 μM, about 500 μM,about 750 μM, or about 1 mM, or a range between any of theaforementioned values.

In one embodiment, the second active agent can be etoposide and theconcentration of etoposide can be about 0.2 μM to about 50 μM.

In another embodiment, the second active agent can be bortezomib and theconcentration of bortezomib can be about 50 nM to about 20 μM.

In another embodiment, the second active agent can be staurosporine andthe concentration of staurosporine can be about 25 nM to about 200 nM.

In another embodiment, the second active agent can be doxorubicin andthe concentration of doxorubicin can be about 50 nM to about 5 μM.

In another embodiment, the second active agent can be tamoxifen and theconcentration of tamoxifen can be about 5 μM to about 50 μM.

In another embodiment, the second active agent can be cisplatin and theconcentration of cisplatin can be about 5 μM to about 150 μM.

In another embodiment, the second active agent can be carboplatin andthe concentration of carboplatin can be about 5 μM to about 150 μM.

In another embodiment, the second active agent can be paclitaxel and theconcentration of paclitaxel can be about 0.5 nM to about 15 nM.

The invention also provides a method of inhibiting the growth orproliferation of cancer cells comprising contacting cancer cells with aneffective amount of a composition of described herein, therebyinhibiting the growth or proliferation of the cancer cells. In someembodiments, the cancer cells can be lymphoma cells, osteosarcoma cells,breast cancer cells, or ovarian cancer cells. In another embodiment, thecancer cells are another cell type described herein below.

The invention further provides a method of inducing apoptosis in acancer cell comprising contacting the cancer cell with an effectiveamount of the compound PAC-1:

-   -   and an effective amount of a second active agent; wherein        apoptosis is thereby induced in the cancer cell. In some        embodiments, the second active agent is etoposide, bortezomib,        staurosporine, doxorubicin, tamoxifen, cisplatin, carboplatin,        or paclitaxel. In other embodiments, the second active agent is        an active agent recited herein below. The contacting can be in        vitro, or the contacting can be in vivo. The cancer cell can be        contacted with PAC-1 and the second active agent concurrently.        Alternatively, the cancer cell can be contacted with PAC-1 prior        to contacting the cancer cell with the second active agent, or        the cancer cell can be contacted with PAC-1 after contacting the        cancer cell with the second active agent.

The invention yet further provides a method of treating a cancer in apatient in need thereof comprising administering to a patient,concurrently or sequentially, a therapeutically effective amount of thecompound PAC-1:

and an effective amount of a second active agent; wherein the cancer isthereby treated. In some embodiments, the second active agent isetoposide, bortezomib, staurosporine, doxorubicin, tamoxifen, cisplatin,carboplatin, or paclitaxel. In other embodiments, the second activeagent is an active agent recited herein below. The compound PAC-1 andthe second active agent can be administered concurrently. Alternatively,the compound PAC-1 and the second active agent can be administeredsequentially. In one embodiment, the compound PAC-lis administeredbefore the second active agent. In another embodiment, the compoundPAC-1 can be administered after the second active agent. The cancer canbe, for example, lymphoma, osteosarcoma, breast cancer, ovarian cancer,or another cancer type recited herein.

The invention thus provides for the use of the compositions describedherein for use in medical therapy. The medical therapy can be treatingcancer, for example, lymphoma, breast cancer, lung cancer, ovariancancer, pancreatic cancer, prostate cancer, colon cancer, and othercancers recited herein. The invention also provides for the use of acomposition as described herein for the manufacture of a medicament totreat a disease in a mammal, for example, cancer in a human. Theinvention thus provides for the use of the compounds described hereinfor the manufacture of medicaments useful for the treatment of cancer ina mammal, such as a human. The medicament can include a pharmaceuticallyacceptable diluent, excipient, or carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1. Chemical structures of structurally diverse chemotherapeuticagents: PAC-1, SPAC-1, etoposide, doxorubicin, bortezomib,staurosporine, and tamoxifen.

FIG. 2. Effects of PAC-1 with Etoposide on U-937 (lymphoma) cell death.Dashed lines represent the level of purely additive effects. The legendcorresponds to the bars of the bar graph as follows: left bar=0 μMetoposide; middle bar=2.5 μM etoposide; right bar=5 etoposide.

FIG. 3. Effects of PAC-1 with Velcade® (bortezomib) on U-937 (lymphoma)cell death. No cell death was observed for 0 nM bortezomib at 0 μMPAC-1. Cell death was measured after 6 hours in bortezomib. Dashed linesrepresent the expected level of purely additive effects; the combinationtherefore shows synergy at therapeutically relevant concentrations.

FIG. 4. Effects of PAC-1 with staurosporine on U-937 (lymphoma) celldeath. Little or no cell death was observed for 0 nM bortezomib at 0-15μM PAC-1. Cell death was measured after 8 hours in staurosporine. Dashedlines represent the expected level of purely additive effects; thecombination therefore shows synergy at therapeutically relevantconcentrations.

FIGS. 5. PAC-1 synergizes with doxorubicin to kill osteosarcoma 143B(human OS) cells. The legend corresponds to the bars of the bar graphwhere the top legend entry corresponds to the left-most bar, and theremaining legend entries correspond to the remaining bars, top to bottomcorresponding to left to right, respectively. No cell death was observedat 0 nM Dox with 0 μM PAC-1. Dashed lines represent the level of purelyadditive effects; the combination therefore shows synergy attherapeutically relevant concentrations.

FIG. 6. PAC-1 potentiates tamoxifen in BT20 (triple negative breastcancer) cells, assessed at 36 hours at various PAC-1 and tamoxifenconcentrations.

FIG. 7. The combination of PAC-1 and tamoxifen is synergistic forkilling BT20 (triple negative breast cancer) cells, assessed at 24 hoursat various PAC-1 and tamoxifen concentrations. The legends correspond tothe bars of the bar graph where the top legend entry corresponds to theleft-most bar, and the remaining legend entries correspond to theremaining bars, top to bottom corresponding to left to right,respectively.

FIG. 8. The combination of PAC-1 and tamoxifen is synergistic forkilling MDA MB 436 (triple negative breast cancer) cells, assessed at 24hours at various PAC-1 and tamoxifen concentrations. The legendscorrespond to the bars of the bar graph where the top legend entrycorresponds to the left-most bar, and the remaining legend entriescorrespond to the remaining bars, top to bottom corresponding to left toright, respectively.

FIG. 9. The combination of PAC-1 and cisplatin is synergistic forkilling IGROV-1 (ovarian carcinoma) cells, assessed at 40 hours (AnnexinV/PI staining) at various PAC-1 and cisplatin concentrations. Thelegends correspond to the bars of the bar graph where the top legendentry corresponds to the left-most bar (absent at 0 μM cisplatin), andthe remaining legend entries correspond to the remaining bars, top tobottom corresponding to left to right, respectively.

FIG. 10. The combination of PAC-1 and paclitaxel is synergistic forkilling IGROV-1 (ovarian carcinoma) cells, assessed at 40 hours (AnnexinV/PI staining) at various PAC-1 and paclitaxel concentrations. Thelegends correspond to the bars of the bar graph where the top legendentry corresponds to the left-most bar (absent at 0 μM paclitaxel), andthe remaining legend entries correspond to the remaining bars, top tobottom corresponding to left to right, respectively.

FIG. 11. PAC-1 synergizes with carboplatin to induce death of HOS (humanosteosarcoma) cells in culture. Cells were co-treated for 8 hours, mediawas replaced and colonies were allowed 7 days to grow.

FIG. 12. PAC-1 synergizes with carboplatin to induce death of 143B(human osteosarcoma) cells in culture. Cells were co-treated for 8hours, media was replaced and colonies were allowed 7 days to grow.

DETAILED DESCRIPTION

As a further introduction, compounds capable of activating an enzymethat is often overexpressed or otherwise present at increased levels inits inactive form in cancer cells have been discovered. The compoundscan induce programmed cell death (apoptosis) in cancer cells, includingthose that have upregulated or increase levels of procaspase-3. Manycancers resist standard chemotherapy. The combination therapy describedherein takes advantage of the procaspase-1 activation by PAC-1, whichcan synergize with the chemotherapeutic properties of a second activeagent, to provide efficacy under conditions where one of the activesalone might be less effective or completely ineffective. These compoundscan also be successful in targeted cancer therapy, where there can beadvantages of selectivity in the killing of cancer cells with comparablyreduced adverse reactions to non-cancerous cells having lower levels ofprocaspase-3. These adverse reactions can include toxicity, particularlyneurotoxicity.

The combination of compounds, compositions and methods described hereincan act via modulation of apoptosis or programmed cell death and otherchemotherapeutic mechanisms to be effective in the treatment of cancercells. In one embodiment, the modulation of apoptosis is by induction oractivation of apoptosis. In various embodiments, the administration ofcompounds can be concurrent, or alternatively, sequential.

The invention thus provides methods for potentiation of an active agentby PAC-1, for example, for the treatment of lymphoma, osteosarcoma, orbreast cancer. During apoptosis, the zymogen procaspase-3 is activatedvia proteolysis to caspase-3, and this active caspase-3 then cleavesscores of cellular substrates, executing the apoptotic program. Becauseprocaspase-3 protein levels are elevated in various tumor histologies,drug-mediated direct activation of procaspase-3 can be highly effectiveas a selective anticancer strategy.

Certain compounds can enhance the activity and automaturation ofprocaspase-3 and induce apoptosis in cancer cells. Procaspase-activatingcompound-1 (PAC-1, FIG. 1) enhances the activity of procaspase-3 via thechelation of inhibitory zinc ions, induces apoptosis in cancer cells inculture, and has efficacy in multiple murine tumor models. Novelcombinations of PAC-1 and several therapeutic agents have been found tobe synergistically effective in treating cancer cells, particularlylymphoma, osteosarcoma, and breast cancer cells, as described herein.Because PAC-1 acts late in the apoptotic cascade, it is uniquely capableof synergizing with a wide range of chemotherapeutic active agents, asdescribed below.

Definitions

As used herein, the recited terms have the following meanings. All otherterms and phrases used in this specification have their ordinarymeanings as one of skill in the art would understand. Such ordinarymeanings may be obtained by reference to technical dictionaries, such asHawley's Condensed Chemical Dictionary 14^(th) Edition, by R. J. Lewis,John Wiley & Sons, New York, N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with the recitation of claim elements or use of a “negative”limitation.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrase “one or more” is readily understood by one of skill in the art,particularly when read in context of its usage. For example, one or moresubstituents on a phenyl ring refers to one to five, or one to four, forexample if the phenyl ring is disubstituted.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percentages, proximate to the recited range that are equivalentin terms of the functionality of the individual ingredient, thecomposition, or the embodiment.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percentages or carbon groups) includes each specific value,integer, decimal, or identity within the range. Any listed range can beeasily recognized as sufficiently describing and enabling the same rangebeing broken down into at least equal halves, thirds, quarters, fifths,or tenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to”, “at least”, “greater than”, “less than”, “more than”,“or more”, and the like, include the number recited and such terms referto ranges that can be subsequently broken down into sub-ranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all sub-ratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, as used in an explicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

“Concurrently” means (1) simultaneously in time, or (2) at differenttimes during the course of a common treatment schedule.

“Sequentially” refers to the administration of one active agent used inthe method followed by administration of another active agent. Afteradministration of one active agent, the next active agent can beadministered substantially immediately after the first, or the nextactive agent can be administered after an effective time period afterthe first active agent; the effective time period is the amount of timegiven for realization of maximum benefit from the administration of thefirst active agent.

An “effective amount” refers to an amount effective to treat a disease,disorder, and/or condition, or to bring about a recited effect, such asactivation or inhibition. For example, an effective amount can be anamount effective to reduce the progression or severity of the conditionor symptoms being treated. Determination of a therapeutically effectiveamount is well within the capacity of persons skilled in the art. Theterm “effective amount” is intended to include an amount of a compounddescribed herein, or an amount of a combination of compounds describedherein, e.g., that is effective to treat or prevent a disease ordisorder, or to treat the symptoms of the disease or disorder, in ahost. Thus, an “effective amount” generally means an amount thatprovides the desired effect. In one embodiment, an effective amountrefers to an amount of the active agent described herein that areeffective, either alone or in combination with a pharmaceutical carrier,upon single- or multiple-dose administration to a cell or a subject,e.g., a patient, at inhibiting the growth or proliferation, inducing thekilling, or preventing the growth of hyperproliferative cells. Suchgrowth inhibition or killing can be reflected as a prolongation of thesurvival of the subject, e.g., a patient beyond that expected in theabsence of such treatment, or any improvement in the prognosis of thesubject relative to the absence of such treatment.

The terms “treating”, “treat” and “treatment” include (i) preventing adisease, pathologic or medical condition from occurring (e.g.,prophylaxis); (ii) inhibiting the disease, pathologic or medicalcondition or arresting its development; (iii) relieving the disease,pathologic or medical condition; and/or (iv) diminishing symptomsassociated with the disease, pathologic or medical condition. Thus, theterms “treat”, “treatment”, and “treating” can extend to prophylaxis andcan include prevent, prevention, preventing, lowering, stopping orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” can include medical, therapeutic,and/or prophylactic administration, as appropriate. In some embodiments,the terms “treatment”, “treat” or “treated” can refer to (i) preventionof tumor growth or regrowth of the tumor (prophylaxis), (ii) a reductionor elimination of symptoms or the disease of interest (therapy) or (iii)the elimination or destruction of the tumor (cure).

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting. Additionally, the terms “induce,” “inhibit,”“potentiate,” “elevate,” “increase,” “decrease,” or the like denotequantitative differences between two states, and can refer to at leaststatistically significant differences between the two states. Forexample, “an amount effective to inhibit the growth ofhyperproliferative cells” means that the rate of growth of the cells canbe, in some embodiments, at least statistically significantly differentfrom the untreated cells. Such terms can be applied herein to, forexample, rates of proliferation.

The phrase “inhibiting the growth or proliferation” of thehyperproliferative cell, e.g. neoplastic cell, refers to the slowing,interrupting, arresting, or stopping its growth and metastasis, and doesnot necessarily indicate a total elimination of the neoplastic growth.

The term “cancer” generally refers to any of a group of more than 100diseases caused by the uncontrolled growth of abnormal cells. Cancer cantake the form of solid tumors and lymphomas, and non-solid cancers suchas leukemia. Unlike normal cells, which reproduce until maturation andthen only as necessary to replace wounded cells, cancer cells can growand divide endlessly, crowding out nearby cells and eventually spreadingto other parts of the body.

The invention provides methods for treating cancer and cancerousconditions. The term “cancerous condition” relates to any conditionwhere cells are in an abnormal state or condition that is characterizedby rapid proliferation or neoplasia. A cancerous condition may bemalignant or non-malignant (e.g. precancerous condition) in nature. Tofarther describe a “cancerous condition”, the terms“hyperproliferative”, “hyperplastic”, “hyperplasia”, “malignant”,“neoplastic” and “neoplasia” can be used. These terms can be usedinterchangeably and are meant to include all types of hyperproliferativegrowth, hyperplastic growth, cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues or organs,irrespective of histopathologic type, stage of invasiveness, orcancerous determination (e.g. malignant and nonmalignant).

The term “neoplasia” refers to new cell growth that results in a loss ofresponsiveness to normal growth controls, e.g., neoplastic cell growth.A “hyperplasia” refers to cells undergoing an abnormally high rate ofgrowth. However, these terms can be used interchangeably, as theircontext will reveal, referring generally to cells experiencing abnormalcell growth rates. “Neoplasias” and “hyperplasias” include tumors, whichmay be either benign, premalignant, carcinoma in-situ, malignant, solidor non-solid. Examples of some cancerous conditions that are within thescope of the invention include, but are not limited to, anal cancer,transitional cell bladder cancer, bone cancer, breast cancer, cervicalcancer, colorectal cancer, gastric cancer, head and neck cancer,Kaposi's sarcoma, leukemia, lung cancer such as bronchogenic lungcancer, small cell lung cancer, and non-small cell lung cancer,Hodgkin's lymphoma, Non-Hodgkin's lymphoma, malignant lymphoma,neuroblastomas, osteogenic carcinomas (e.g. cancer of the bone),ophthalmic cancers (e.g. retinoblastomas and other cancers of the eye),ovarian cancer, prostate cancer, renal cancer, skin cancers such asmelanoma, soft tissue sarcomas, thyroid cancer, and Wilms' tumor. Otherexamples of non-malignant hyperproliferative conditions (e.g.precancerous conditions) that are within the scope of the inventioninclude, but are not limited to, adenomas, chondromas, enchondromas,fibromas, myomas, myxomas, neurinomas, osteoblastomas, osteochondromas,osteomas, papillary tumors, and the like.

The terms “leukemia” or “leukemic cancer” refer to all cancers orneoplasias of the hematopoetic and immune systems (blood and lymphaticsystem). These terms refer to a progressive, malignant disease of theblood-forming organs, marked by distorted proliferation and developmentof leukocytes and their precursors in the blood and bone marrow.Myelomas refer to other types of tumors of the blood and bone marrowcells. Lymphomas refer to tumors of the lymph tissue. Examples ofleukemia include acute myelogenous leukemia (AML), acute lymphoblasticleukemia (ALL), and chronic myelogenous leukemia (CIVIL).

As described herein, the compositions and methods of the invention canbe used for the treatment or prevention of various neoplasia disordersincluding such conditions as acral lentiginous melanoma, actinickeratoses, adenocarcinoma, adenoid cycstic carcinoma, adenomas,adenosarcoma, adenosquamous carcinoma, astrocytic tumors, bartholingland carcinoma, basal cell carcinoma, bronchial gland carcinomas,capillary, carcinoids, carcinoma, carcinosarcoma, cavernous,cholangiocarcinoma, chondosarcoma, choriod plexus papilloma/carcinoma,clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrialhyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma,ependymal, epitheloid, Ewing's sarcoma, fibrolamellar, focal nodularhyperplasia, gastrinoma, germ cell tumors, glioblastoma, glucagonoma,hemangiblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma,hepatic adenomatosis, hepatocellular carcinoma, insulinoma,intaepithelial neoplasia, interepithelial squamous cell neoplasia,invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma,lentigo maligna melanomas, malignant melanoma, malignant mesothelialtumors, medulloblastoma, medulloepithelioma, melanoma, meningeal,mesothelial, metastatic carcinoma, mucoepidermoid carcinoma,neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, oat cellcarcinoma, oligodendroglial, osteosarcoma, pancreatic polypeptide,papillary serous adenocarcinoma, pineal cell, pituitary tumors,plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cellcarcinoma, soft tissue carcinomas, somatostatin-secreting tumor,squamous carcinoma, squamous cell carcinoma, submesothelial, superficialspreading melanoma, undifferentiated carcinoma, uveal melanoma,verrucous carcinoma, vipoma, well differentiated carcinoma, and Wilm'stumor. Accordingly, the compositions and methods described herein can beused to treat bladder cancer, brain cancer (including intracranialneoplasms such as glioma, meninigioma, neurinoma, and adenoma), breastcancer, colon cancer, lung cancer (SCLC or NSCLC) ovarian cancer,pancreatic cancer, and prostate cancer.

In some embodiments, the combination of PAC-1 and a second active agent(e.g., a chemotherapeutic agent recited herein) can be particularlyeffective for treating cancers of the brain. Cancers of the braininclude, but are not limited to, oligodendrogliomas and glioblastomasincluding glioblastoma multiforme (GBM). Tissues affected by thecancerous cells can be in the brain itself (e.g., the cranium or thecentral spinal canal) or in lymphatic tissue, in blood vessels, in thecranial nerves, in the brain envelopes (meninges), skull, pituitarygland, or pineal gland. Specific forms of brain cancer that can betreated include astrocytomas, chondromas, chondrosarcomas, chordomas,CNS (central nervous system) lymphomas, craniopharyngiomas, ependymomas,gangliogliomas, ganglioneuromas (also called gangliocytomas), gliomas,including astrocytomas, oligodendrogliomas, and ependymomas,hemangioblastomas (also called vascular tumors), primitiveneuroectodermal tumors (PNET) such as medulloblastomas, meningiomas, andvestibular schwannomas (formerly known as acoustic neuroma/schwannoma).

The combination can also be used to treat metastatic tumors that invadethe intracranial sphere from cancers originating in other organs of thebody. These conditions are typically referred to as secondary braintumors. Secondary brain tumors that can be treated with the combinationof PAC-1 and a second active agent include metastatic tumors of thebrain that originate from lung cancer, breast cancer, malignantmelanoma, kidney cancer, colon cancer, and other carcinomas.

Other examples of cancerous conditions that are within the scope of theinvention include, but are not limited to, neuroblastomas and osteogeniccarcinomas (e.g. cancer of the bone or neoplastic growth of tissue inbone). Examples of malignant primary bone tumors that can be treatedwith the combination of PAC-1 and a second active agent includeosteosarcomas, chondrosarcomas, Ewing's sarcoma, fibrosarcomas, and thelike, and secondary bone tumors such as metastatic lesions that havespread from other organs, including carcinomas of the breast, lung, andprostate.

Therapeutic Agents and Activity

Procaspase-activating compound-1 (PAC-1;(2-(4-benzylpiperazin-1-yl)-N-[(2-hydroxy-3-prop-2-enyl-phenyl)methylideneamino]acetamide)selectively induces apoptosis in cancerous cells. The structure of PAC-1is shown in FIG. 1 and methods of preparing PAC-1 are described in U.S.Patent Publication No. 2012/0040995 (Hergenrother et al.).

PAC-1 enhances the activity of procaspase-3 via the chelation ofinhibitory zinc ions, induces apoptosis in cancer cells. PAC-1 canenhance the activity and automaturation of procaspase-3 and induceapoptosis in cancer cells. PAC-1 also enhanced that chemotherapeuticactivity of several other drugs, often where either PAC-1 or the secondactive is less effective or completely inactive alone. Accordingly, itwas surprisingly discovered that PAC-1 synergizes the activity ofnumerous classes of chemotherapeutic agents. Examples of classes ofcompounds that can synergize with PAC-1 include:

-   -   (a) bcl-2 family inhibitors/modulators (including bax and bcl-xl        inhibitors);    -   (b) modulators of BIR motif containing proteins (e.g., survivin,        SMAC mimetics, and the like);    -   (c) modulators/stabilizers or inhibitors of microtubules or        cytoskeletal elements (e.g., taxanes such as paclitaxel and        docetaxel);    -   (d) alkylating agents such as cyclophosphamide, DTIC or        cytotoxic antibiotics such as doxorubicin;    -   (e) DNA intercalating agents (e.g. platins such as cisplatin,        carboplatin or oxaliplatin);    -   (f) autophagy modulating agents such as temozolomide;    -   (g) tumor cell signal transduction inhibitors (e.g. inhibitors        or wild type or mutant EGFRs, braf, Ras, AKT, cMET, mTOR, PI3K,        BTK, JAK/STAT family members, MEK);    -   (h) inhibitors/modulators of signaling receptors (e.g.        tamoxifen, antibodies to EGFRs, CD20, CD19, and others over        expressed or routinely expressed on tumor cells);    -   (i) inhibitors/modulators of angiogenesis (e.g., VEGFs, VEGFRs,        angiogenins, angiostatins, TIE proteins, endostatins, and the        like);    -   (j) modulators of immune mediated mechanism (e.g., vaccines,        cell therapies, checkpoint inhibitors, pro-inflammatory        cytokines/antibodies, adjuvants, and the like); and    -   (k) proteasome inhibitors such as bortezomib.

Examples of specific chemotherapeutic agents (active agents, or ‘secondactive agents’) and can advantageously combined with PAC-1 includeactive agents such as, cisplatin, etoposide, irinotecan, camptostar,topotecan, paclitaxel, docetaxel, epothilones, taxotere, tamoxifen,5-fluorouracil, methoxtrexate, temozolomide, cyclophosphamide, SCH66336, R115777, L778,123, BMS 214662, gefitinib, erlotinibhydrochloride, antibodies to EGFR, imatinib, intron, ara-C, cytoxan,gemcitabine, uracil mustard, chlormethine, ifosfamide, melphalan,chlorambucil, pipobroman, triethylenemelamine,triethylenethiophosphoramine, busulfan, carmustine, lomustine,streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine,6-thioguanine, fludarabine phosphate, pentostatine, vinblastine,vincristine, vindesine, bleomycin, doxorubicin, dactinomycin,daunorubicin, epirubicin, idarubicin, mithramycin, deoxycoformycin,L-asparaginase, teniposide, ethinyl estradiol, diethylstilbestrol,testosterone, prednisone, fluoxymesterone, dromostanolone propionate,testolactone, megestrolacetate, methylprednisolone, methyltestosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesteroneacetate, leuprolide,flutamide, toremifene, goserelin, hydroxyurea, amsacrine, procarbazine,mitotane, mitoxantrone, levamisole, navelbene, anastrazole, letrazole,capecitabine, reloxafine, droloxafine, hexamethylmelamine, bevacizumab,herceptin, Bexxar, Zevalin, Trisenox, Xeloda, Vinorelbine, Porfimer,cetuximab, Thiotepa, Altretamine, Melphalan, Trastuzumab, Lerozole,Fulvestrant, Exemestane, Fulvestrant, Ifosfomide, Rituximab, C225,Campath, carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, busulfan, nitrosurea, plicomycin, mitomycin, raloxifene,estrogen receptor binding agents, navelbine, farnesyl-proteintransferase inhibitors, transplatinum and methotrexate, or any analog orderivative variant of the foregoing.

Examples of chemotherapeutic active agent that show significant activitywhen combined with PAC-1 or a PAC-1 derivative include etoposide,bortezomib, staurosporine, doxorubicin, tamoxifen, cisplatin,carboplatin, paclitaxel, and SMAC mimetic.

Combination with Etoposide

Etoposide is a topoisomerase II inhibitor. Etoposide forms a ternarycomplex with DNA and the topoisomerase II enzyme, preventing re-ligationof the DNA strands, which causes errors in DNA synthesis and promotesapoptosis of the cancer cell. Combined treatment of U-937 cells withPAC-1 and etoposide showed significant in vitro activity even at lowmicromolar concentrations (FIG. 2).

Combination with Bortezomib

Velcade® (bortezomib) binds the catalytic site of the 26S proteasomewith high affinity and specificity. In normal cells, the proteasomeregulates protein expression and function by degradation ofubiquitinylated proteins, and also cleanses the cell of abnormal ormisfolded proteins. While multiple mechanisms are likely to be involved,proteasome inhibition may prevent degradation of pro-apoptotic factors,permitting activation of programmed cell death in neoplastic cellsdependent upon suppression of pro-apoptotic pathways. Synergisticactivity was observed for the combination treatment of U-937 lymphomacells with PAC-1 and bortezomib (FIG. 3).

Combination with Staurosporine

The main biological activity of staurosporine is the inhibition ofprotein kinases through the prevention of ATP binding to the kinase,which is achieved through the stronger affinity of staurosporine to theATP-binding site on the kinase. Staurosporine is a prototypicalATP-competitive kinase inhibitor in that it binds to many kinases withhigh affinity, though with low selectivity. The lack of specificity hasprecluded its clinical use but has made it a valuable research toolwhere staurosporine is used to induce apoptosis. One way thatstaurosporine induces apoptosis is by activating caspase-3. Combinedtreatment of U-937 lymphoma cells with PAC-1 and staurosporine showedsynergistic effects at low PAC-1 concentrations, such as at 7.5 μM and15 μM PAC-1 (FIG. 4).

Combination with Doxorubicin

Doxorubicin is an anthracycline antibiotic that exerts its cytotoxicactivity by DNA intercalation. Doxorubicin is used to treat a wide rangeof cancers, including hematological malignancies, many types ofcarcinoma, and soft tissue sarcomas and osteosarcomas. Synergisticactivity was observed for the combination treatment of 143B (human OS)osteosarcomas cells with PAC-1 and doxorubicin (FIG. 5).

Combination with Tamoxifen

Tamoxifen, a competitive agonist of estrogen receptor is the most commontreatment for male breast cancer and is used for both early and advancedER+ breast cancers. Tamoxifen is approved for the prevention of breastcancer in those at high risk. The combination of tamoxifen and PAC-1 issynergistic and provides enhanced cell killing efficiencies in breastcancer, including tamoxifen negative or tamoxifen resistant breastcancer, and triple negative breast cancer (FIGS. 6-8).

Combination with Cisplatin

Cisplatin is one of several platinum coordination complexes that areused in cancer chemotherapy. The cytotoxicity of platinum compounds canresult from inhibition of DNA synthesis in cancer cells. Cisplatin isused for the treatment of various types of cancers, including sarcomas,carcinomas (including small cell lung cancer and ovarian cancer),lymphomas, germ cell tumors, and testicular cancer. The combination ofcisplatin and PAC-1 can be synergistic and can provide enhanced cellkilling efficiencies in these treatments as well as ovarian carcinoma(FIG. 9).

Combination with Paclitaxel

Paclitaxel, a mitotic inhibitor (microtubule stabilizer), is used in thetreatment of lung, ovarian, breast, and head and neck cancer. Paclitaxelis recommended for the treatment of advanced breast cancer after failureof anthrocyclines and is recommended against use in early node-positivebreast cancer. The combination of paclitaxel and PAC-1 providessynergistic activity and enhanced cell killing efficiencies in thesetreatments as well as ovarian carcinoma (FIG. 10).

Combination with Carboplatin

Carboplatin is another one of the several platinum coordinationcomplexes that are used in cancer chemotherapy. Carboplatin is used forthe treatment of various types of cancers, mainly ovarian carcinoma,lung, head and neck cancers. The combination of carboplatin and PAC-1 issynergistic and can provide enhanced cell killing efficiencies in thesetreatments as well as osteosarcoma (FIGS. 11 and 12).

Further Combination Studies

Utilizing cell lines that represent of 12 of the 17 recently definedbreast cancer subtypes (Table 1), examination of nonlethal doses ofPAC-1 sensitization/synergy with standard of care drugs is underway.

TABLE 1 Breast cancer cell lines under investigation. Subgroup Cell Line5 BT20 5 BT549 5 Hs578T 2 HCC1569 4 MCF7 4 T47D 6 MDAMB361 7 AU565 8HCC1954 9 MDAMB231 10 HCC202 14 MDAMB436 16 BT483

PAC-1 combined with a variety of different standard of care agents canalso provide additive or synergistic activity that may not be otherwiseobtainable. Examples of such standard of care agents being investigatedfor combination effects include:

Lapatinib, a dual tyrosine kinase inhibitor (EGFR and HER2) is used intherapy for HER2 positive cancers and front line therapy for triplepositive breast cancers. The combination of lapatinib and PAC-1 canprovide enhanced cell killing efficiencies in these treatments.

Fluorouracil (5-FU) is a pyrimidine analog drug that is used in thetreatment of a variety of cancers. It is a suicide inhibitor and worksthrough irreversible inhibition of thymidylate synthase. The combinationof 5-FU and PAC-1 can provide enhanced cell killing efficiencies incancers treatable by 5-FU.

In various embodiments, PAC-1 can be exchanged with its analog SPAC-1for similar enhanced, additive, or synergistic activity. The combinationeffects of SPAC-1 with common oncological therapies against colon, lungand liver cancer cell lines are being investigated. Examples of thecombination agents, cell lines and data output that can be obtained aresummarized in Table 2 and Table 3, where, for example, PAC-1 or SPAC-1can be combined with any of Standard Agents 1-4.

TABLE 2 Cell lines and agents for Combination effects. Cell Line TissueStd. Agent 1 Std. Agent 2 Std. Agent 3 Std Agent 4 DLD-1 (BIRC5↑) ColonSN-38 Oxaliplatin HCT-116 Colon SN-38 Oxaliplatin Hep3B Liver SorafenibSunitinib HepG2 Liver Sorafenib Sunitinib A549 (NSCL-AC) LungOxaliplatin Gemcitabine Erlotinib Pemetrexed H292 (NSCL-C) LungOxaliplatin Gemcitabine Erlotinib Pemetrexed SK-MES-1 (SCC) LungOxaliplatin Etoposide Gemcitabine

TABLE 3 Data under investigation for combination effect experiments,where Drug 1 is PAC-1 or a derivative thereof and Drug 2 is an activeagent recited or described herein. Drug 1 0 0.25X (IC₅₀) 0.5X (IC₅₀)1.0X (IC₅₀) 2.0X (IC₅₀) 4.0X (IC₅₀) Drug 2 0 Control (F_(a))₁ (F_(a))₁(F_(a))₁ (F_(a))₁ (F_(a))₁ 0.25X (IC₅₀) (F_(a))₂ (F_(a))_(1, 2) 0.5X(IC₅₀) (F_(a))₂ (F_(a))_(1, 2) 1.0X (IC₅₀) (F_(a))₂ (F_(a))_(1, 2) 2.0X(IC₅₀) (F_(a))₂ (F_(a))_(1, 2) 4.0X (IC₅₀) (F_(a))₂ (F_(a))_(1, 2) F_(a)= Fraction of cell affected by treatment

Active agents that can be combined with PAC-1 or a derivative thereof toprovide enhanced or synergistic activity for inhibiting cancer cellgrowth or for treating a particular type of cancer further include:

SN-38 is the active metabolite of irinotecan (an analog of camptothecin,a topoisomerase I inhibitor). SN-38 is 200 times more active thanirinotecan itself. Irinotecan's main use is in colon cancer, inparticular, in combination with other chemotherapy agents. Thecombination of SN-38 and PAC-1 can provide enhanced cell killingefficiencies in these treatments.

Oxaliplatin is one of several platinum coordination complexes that areused in cancer chemotherapy. The cytotoxicity of platinum compounds isthought to result from inhibition of DNA synthesis in cancer cells. Invivo studies showed that oxaliplatin has anti-tumor activity againstcolon carcinoma through its (non-targeted) cytotoxic effects. Thecombination of oxaliplatin and PAC-1 can provide enhanced cell killingefficiencies in these treatments.

Sorafenib is a small molecular inhibitor of several Tyrosine proteinkinases (VEGFR and PDGFR) and Raf. Sorafenib targets the MAP kinasepathway (Raf/Mek/Erk pathway) (MAP Kinase pathway) and is approved forthe treatment of primary kidney cancer (advanced renal cell carcinoma)and advanced primary liver cancer (hepatocellular carcinoma). Thecombination of sorafenib and PAC-1 can provide enhanced cell killingefficiencies in these treatments.

Sunitinib is an oral, small-molecule, multi-targeted receptor tyrosinekinase (RTK) inhibitor that was approved by the FDA for the treatment ofrenal cell carcinoma (RCC) and imatinib-resistant gastrointestinalstromal tumor (GIST). The combination of sunitinib and PAC-1 can provideenhanced cell killing efficiencies in these treatments.

Gemcitabine is a nucleoside analog used for chemotherapy. As withfluorouracil and other analogues of pyrimidines, the triphosphateanalogue of gemcitabine replaces one of the building blocks of nucleicacids, in this case cytidine, during DNA replication. The processarrests tumor growth, as only one additional nucleoside can be attachedto the “faulty” nucleoside, resulting in apoptosis. Another target ofgemcitabine is the enzyme ribonucleotide reductase (RNR). Thediphosphate analogue binds to the RNR active site and inactivates theenzyme irreversibly. Once RNR is inhibited, the cell cannot produce thedeoxyribonucleotides required for DNA replication and repair, and cellapoptosis is induced. The combination of gemcitabine and PAC-1 canprovide enhanced cell killing efficiencies in these treatments.

Erlotinib is a drug used to treat non-small cell lung cancer, pancreaticcancer and several other types of cancer. It is a tyrosine kinaseinhibitor, which acts on the epidermal growth factor receptor (EGFR).The combination of erlotinib and PAC-1 can provide enhanced cell killingefficiencies in these treatments.

Pemetrexed is a chemotherapy drug used in the treatment of pleuralmesothelioma as well as non-small cell lung cancer. Pemetrexed is in theclass of chemotherapy drugs called folate antimetabolites. It works byinhibiting three enzymes used in purine and pyrimidinesynthesis-thymidylate synthase (TS), dihydrofolate reductase (DHFR), andglycinamide ribonucleotide formyltransferase (GARFT). By inhibiting theformation of precursor purine and pyrimidine nucleotides, pemetrexedprevents the formation of DNA and RNA, which are required for the growthand survival of both normal cells and cancer cells. The combination ofpemetrexed and PAC-1 can provide enhanced cell killing efficiencies inthese treatments.

While there is clear benefit to anticancer strategies utilizingcombinations of drugs that act on different targets, the work describedherein demonstrates that dramatic synergy can be observed with compoundsthat act through disparate mechanisms. This multi-targeting approach canhave particular advantages when activation of an enzyme is sought.

PAC-1 is safe in mammals, and a derivative of PAC-1 was efficacious in aphase I clinical trial of pet dogs with lymphoma (Peterson et al.,Cancer Res 70, 7232-7241 (2010)), thus the observed synergy with activeagents such as etoposide, bortezomib, staurosporine, doxorubicin, andtamoxifen will have significant clinical impact. Interest in activatingenzymes with small molecules is increasing rapidly. The data describedherein indicate that targeting strategies using PAC-1 and suchcomplimentary active agents is a general approach for dramaticenhancement of the intended biologic effect and should have considerableclinical impact due to its efficacy.

Methods of the Invention

The invention provides methods of selectively inducing apoptosis in acancer cell, comprising administering to a cancer cell a combination ofcompounds capable of modifying a procaspase-3 molecule of said cancercell; wherein the combination of compounds is PAC-1 and a second activeagent. Also provided is a method of selectively inducing apoptosis in acancer cell, comprising administering to a cancer cell a combination ofcompounds capable of modifying a procaspase-3 molecule of the cancercell; wherein the combination of compounds is PAC-1 and a second activeagent, for example, wherein the cancer cell is in a patient in need oftreatment.

The invention provides additional methods where the recited combinationof compounds is PAC-1 and a second active agent, for example, as amethod of treating a cancer cell, comprising (a) identifying a potentialsusceptibility to treatment of a cancer cell with a procaspase activatorcompound; and (b) exposing the cancer cell to an effective amount of acombination of a procaspase activator compound and a second activeagent. Also provided is a method of treating a cancer cell, comprising(a) identifying a potential susceptibility to treatment of a cancer cellwith a procaspase activator compound; and (b) exposing said cancer cellto an effective amount of PAC-1 and a second active agent; wherein thePAC-1 is capable of activating at least one of procaspase-3 andprocaspase-7. Also provided is a method of inducing death in a cancercell (e.g., killing a cancer cell), comprising administering to a cancercell an active agent and a compound capable of activating a procaspase-3molecule of the cancer cell, such as PAC-1.

The invention further provides a medicament comprising an effectiveamount of the combination of PAC-1 and a second active agent. Themedicament can be used in a method of inducing apoptosis in a cell. Insome embodiments, the combination of compounds does not cross theblood-brain barrier to as extent that causes appreciable neurotoxiceffects in a patient. Methods of the invention include contacting one ormore cells with an effective amount of a combination of compoundsdescribed herein, in vivo or in vitro. The invention thus also providesmethods of treating a cell that include contacting a cell with aneffective amount of a combination of compounds described herein.

As described herein, the invention provides methods of treating apatient that has tumor cells having elevated procaspase-3 levels. Themethods can include administering to a patient having tumor cells withelevated procaspase-3 levels a therapeutically effective amount of acombination of PAC-1 and a second active agent described herein, or acomposition thereof. The invention further provides methods of treatinga tumor cell having an elevated procaspase-3 level comprising exposingthe tumor cell to a therapeutically effective amount of a combination ofPAC-1 and a second active agent described herein, wherein the tumor cellis treated, killed, or inhibited from growing. The tumor or tumor cellscan be malignant tumor cells. In some embodiments, the tumor cells arelymphoma, osteosarcoma, or breast cancer cells.

PAC-1 can be combined with a second active agent in a unitary dosageform for the administration to a patient. The combination therapy may beadministered as a simultaneous or sequential regimen. When administeredsequentially, the combination may be administered in two or moreadministrations.

The combination therapy may provide “synergy”, i.e. the effect achievedwhen the active ingredients used together is greater than the sum of theeffects that results from using the compounds separately. A synergisticeffect may be attained when PAC-1 and a second active agent are: (1)co-formulated and administered or delivered simultaneously in a combinedformulation; (2) delivered by alternation or in parallel as separateformulations; or (3) by some other regimen. When delivered inalternation therapy, a synergistic effect may be attained when thecompounds are administered or delivered sequentially, e.g. in separatetablets, pills or capsules, or by different injections in separatesyringes. In general, during alternation therapy, an effective dosage ofeach active ingredient can be administered sequentially, i.e. serially,whereas in combination therapy, effective dosages of two or more activeingredients are administered together. A synergistic anti-cancer effectdenotes an anti-cancer effect that is greater than the predicted purelyadditive effects of the individual compounds of the combination.Combination therapy is further described by U.S. Pat. No. 6,833,373(McKearn et al.), which includes additional active agents that can becombined with PAC-1, and additional types of cancer and other conditionsthat can be treated with PAC-1.

Accordingly, PAC-1 can be used in combination with another active agent(“a second active agent”) for cancer treatment. PAC-1 may precede orfollow the second active agent administration by intervals ranging fromminutes to weeks. In embodiments where the second active agent and PAC-1are applied separately to the cell, one would generally ensure that asignificant period of time did not elapse between the time of eachdelivery, such that the agent and PAC-1 would still be able to exert anadvantageously combined effect on the cell. For example, in suchinstances, it is contemplated that one may contact the cell, tissue ororganism with the two modalities substantially simultaneously (i.e.,within less than about a few minutes). In other aspects, the secondactive agent of the combination may be administered within about 1minute, about 5 minutes, about 10 minutes, about 20 minutes about 30minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3hours, about 4 hours, about 6 hours, about 8 hours, about 9 hours, about12 hours, about 15 hours, about 18 hours, about 21 hours, about 24hours, about 28 hours, about 31 hours, about 35 hours, about 38 hours,about 42 hours, about 45 hours, or at about 48 hours or more, prior toand/or after administering PAC-1. In certain other embodiments, thesecond active agent may be administered within about 1 day, about 2days, about 3 days, about 4 days, about 5 days, about 6 days, about 8days, about 9 days, about 12 days, about 15 days, about 16 days, about18 days, about 20 days, or about 21 days, prior to and/or afteradministering PAC-1. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where severalweeks (e.g., about 1, about 2, about 3, about 4, about 6, or about 8weeks or more) lapse between the respective administrations.

Administration of the chemotherapeutic compositions of the invention toa patient will typically follow general protocols for the administrationof chemotherapeutics, taking into account the toxicity, if any. It isexpected that the treatment cycles would be repeated as necessary. Italso is contemplated that various standard therapies or adjunct cancertherapies, as well as surgical intervention, may be applied incombination with the described combinations. These therapies include butare not limited to chemotherapy, immunotherapy, gene therapy andsurgery.

Pharmaceutical Formulations

The compounds described herein can be used to prepare therapeuticpharmaceutical compositions, for example, by combining the compoundswith a pharmaceutically acceptable diluent, excipient, or carrier. Thecompounds may be added to a carrier in the form of a salt or solvate.For example, in cases where compounds are sufficiently basic or acidicto form stable nontoxic acid or base salts, administration of thecompounds as salts may be appropriate. Examples of pharmaceuticallyacceptable salts are organic acid addition salts formed with acids thatform a physiological acceptable anion, for example, tosylate,methanesulfonate, acetate, citrate, malonate, tartrate, succinate,benzoate, ascorbate, α-ketoglutarate, and β-glycerophosphate. Suitableinorganic salts may also be formed, including hydrochloride, halide,sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid to provide aphysiologically acceptable ionic compound. Alkali metal (for example,sodium, potassium or lithium) or alkaline earth metal (for example,calcium) salts of carboxylic acids can also be prepared by analogousmethods.

The compounds described herein can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient, in a variety of forms. The forms can be specifically adapted toa chosen route of administration, e.g., oral or parenteraladministration, by intravenous, intramuscular, topical or subcutaneousroutes.

The compounds described herein may be systemically administered incombination with a pharmaceutically acceptable vehicle, such as an inertdiluent or an assimilable edible carrier. The solubility of actives canbe increase by the use of cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin. For oral administration, compounds canbe enclosed in hard or soft shell gelatin capsules, compressed intotablets, or incorporated directly into the food of a patient's diet.Compounds may also be combined with one or more excipients and used inthe form of ingestible tablets, buccal tablets, troches, capsules,elixirs, suspensions, syrups, wafers, and the like. Such compositionsand preparations typically contain at least 0.1% of active compound. Thepercentage of the compositions and preparations can vary and mayconveniently be from about 1% to about 60%, or about 2% to about 25%, ofthe weight of a given unit dosage form. The amount of active compound insuch therapeutically useful compositions is such that an effectivedosage level can be obtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. A sweeteningagent such as sucrose, fructose, lactose or aspartame; or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring, maybe added. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye andflavoring such as cherry or orange flavor. Any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations anddevices.

The active compound may be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can be prepared in glycerol, liquidpolyethylene glycols, triacetin, or mixtures thereof, or in apharmaceutically acceptable oil. Under ordinary conditions of storageand use, preparations may contain a preservative to prevent the growthof microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions, dispersions, or sterile powderscomprising the active ingredient adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. The ultimate dosage form should besterile, fluid and stable under the conditions of manufacture andstorage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), vegetable oils, nontoxic glyceryl esters, and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe formation of liposomes, by the maintenance of the required particlesize in the case of dispersions, or by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thiomersal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by agents delayingabsorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, optionally followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, methods of preparation can includevacuum drying and freeze drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

Useful dosages of the compounds described herein can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949 (Borch et al.). The amount of a compound, or anactive salt or derivative thereof, required for use in treatment willvary not only with the particular compound or salt selected but alsowith the route of administration, the nature of the condition beingtreated, and the age and condition of the patient, and will beultimately at the discretion of an attendant physician or clinician.

The combination of compounds can be conveniently administered in a unitdosage form, for example, containing 100 to 5,000 mg/m², 300 to 4,000mg/m², 370 to 3,700 mg/m², 50 to 750 mg/m², or 750 to 4,000 mg/m² ofactive ingredient per unit dosage form. Each compound, individually orin combination, can also be administered at about 1 mg/kg to about 250mg/kg, about 10 mg/kg to about 100 mg/kg, about 10 mg/kg to about 50mg/kg, about 50 mg/kg to about 100 mg/kg, about 10 mg/kg to about 50mg/kg, or about 10 mg/kg, about 25 mg/kg, about 50 mg/kg, about 75mg/kg, about 100 mg/kg, or about 150 mg/kg, or a range from any one ofthe aforementioned values to any other of the aforementioned values. Thecompounds can also be administered to a subject to provide asteady-state plasma concentration of the drugs, alone or in combination,of about 1 μmon to about 25 μmon, or about 10 μmon, or about 15 μmon.

In some embodiments, the invention provides the compounds in effectiveconcentrations at about 10 nM to about 100 μM. In another embodiment,the effective concentrations are from about 200 nM to about 50 μM, about500 nM to about 40 μM, about 750 nM to about 25 μM, about 1 μM to about20 μM, or about 1 μM to about 10 μM. In another embodiment, theeffective concentration is considered to be a value such as a 50%activity concentration in a direct procaspase activation assay, in acell apoptosis induction assay, or in an animal clinical therapeuticassessment. In one embodiment, such value is less than about 200 μM. Inanother embodiment, the value is less than about 10 μM but greater thanabout 10 nM. The desired dose may conveniently be presented in a singledose or as divided doses administered at appropriate intervals, forexample, as two, three, four or more sub-doses per day. The sub-doseitself may be further divided, e.g., into a number of discrete looselyspaced administrations.

The compounds described herein can be effective anti-tumor agents andhave higher potency and/or reduced toxicity as compared to theadministration of any single agent. The invention provides therapeuticmethods of treating cancer in a mammal, which involve administering to amammal having cancer an effective amount of a compound or compositiondescribed herein. A mammal includes a primate, human, rodent, canine,feline, bovine, ovine, equine, swine, caprine, bovine and the like.Cancer refers to any various type of malignant neoplasm, for example,colon cancer, breast cancer, melanoma and leukemia, among othersdescribed herein, and in general is characterized by an undesirablecellular proliferation, e.g., unregulated growth, lack ofdifferentiation, local tissue invasion, and metastasis.

The ability of a compound of the invention to treat cancer may bedetermined by using assays well known to the art. For example, thedesign of treatment protocols, toxicity evaluation, data analysis,quantification of tumor cell kill, and the biological significance ofthe use of transplantable tumor screens are known. In addition, abilityof a compound to treat cancer may be determined using the assaysdescribed above and in the citations and patent documents cited herein.

The invention also provides prodrug forms of compounds. Any compoundthat will be converted in vivo to provide PAC-1 or another active agentrecited herein is a prodrug. Numerous methods of forming prodrugs arewell known in the art. Examples of prodrugs and methods of preparingthem are found, inter alia, in Design of Prodrugs, edited by H.Bundgaard, (Elsevier, 1985), Methods in Enzymology, Vol. 42, at pp.309-396, edited by K. Widder, et al. (Academic Press, 1985); A Textbookof Drug Design and Development, edited by Krosgaard-Larsen and H.Bundgaard, Chapter 5, “Design and Application of Prodrugs,” by H.Bundgaard, at pp. 113-191, 1991); H. Bundgaard, Advanced Drug DeliveryReviews, Vol. 8, p. 1-38 (1992); H. Bundgaard, et al., J. Pharm. Sci.,Vol. 77, p. 285 (1988); and Nogrady (1985) Medicinal Chemistry ABiochemical Approach, Oxford University Press, New York, pages 388-392).

Additionally, in some embodiments, PAC-1 can be exchanged for a PAC-1derivative or other inhibitor, such as a compound described in U.S. Pat.No. 7,632,972 (Hergenrother et al.), U.S. Patent Publication Nos.2012/0040995 (Hergenrother et al.) and 2007/0049602 (Hergenrother etal.), and U.S. application Ser. No. 12/597,287 (Hergenrother et al.).Useful compounds, methods, and techniques for cancer therapy that can beused in combination with the disclosure herein are described in theaforementioned documents, as well as in U.S. Pat. No. 6,303,329(Heinrikson et al.), U.S. Pat. No. 6,403,765 (Alnemri), U.S. Pat. No.6,878,743 (Choong et al.), and U.S. Pat. No. 7,041,784 (Wang et al.),and U.S. Patent Publication No. 2004/0180828 (Shi).

Methods for performing the tests and evaluating cancer cell lines can becarried out as described by Putt et al., Nature Chemical Biology 2006,2(10), 543-550; Peterson et al., J. Mol. Biol. 2009, 388, 144-158; andPeterson et al., Cancer Res. 2010, 70(18), 7232-7241.

The following Example is intended to illustrate the above invention andshould not be construed as to narrow its scope. One skilled in the artwill readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

EXAMPLES Example 1. Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of the combination compounds described herein (e.g.,PAC-1 and the second active agent), or pharmaceutically acceptable saltsor solvates thereof (hereinafter referred to as ‘Compounds X’):

(i) Tablet 1 mg/tablet ‘Compounds X’ 200.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 400.0 (ii) Tablet 2 mg/tablet ‘Compounds X’ 120.0Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate15.0 Magnesium stearate 5.0 600.0 (iii) Capsule mg/capsule ‘Compounds X’110.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinized starch120.0 Magnesium stearate 3.0 700.0 (iv) Injection 1 (1 mg/mL) mg/mL‘Compounds X’ 1.0 Dibasic sodium phosphate 12.0 Monobasic sodiumphosphate 0.7 Sodium chloride 4.5 1.0N Sodium hydroxide solution q.s.(pH adjustment to 7.0-7.5) Water for injection q.s. ad 1 mL (v)Injection 2 (10 mg/mL) mg/mL ‘Compounds X’ 10.0 Monobasic sodiumphosphate 0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.00.1N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water forinjection q.s. ad 1 mL (vi) Aerosol mg/can ‘Compounds X’ 20 Oleic acid10 Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Compounds X’. Aerosol formulation (vi) may be used inconjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Nolimitations inconsistent with this disclosure are to be understoodtherefrom. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

What is claimed is:
 1. A method of treating cancer in a subject in needthereof comprising administering to a subject in need of therapy forcancer, concurrently or sequentially, a therapeutically effective amountof the compound PAC-1

and an effective amount of a second active agent, wherein the secondactive agent is a topoisomerase inhibitor, a DNA intercalating agent, amitotic inhibitor, a proteasome inhibitor, a protein kinase inhibitor,or a competitive agonist of the estrogen receptor; wherein the cancer isthereby treated.
 2. The method of claim 1 wherein the topoisomeraseinhibitor is doxorubicin, irinotecan, or SN-38.
 3. The method of claim 1wherein the DNA intercalating agent is cisplatin, carboplatin,oxaliplatin, mitomycin, carmustine, 5-fluorouricil, gemcitabine, orchlorambucil.
 4. The method of claim 1 wherein the mitotic inhibitor ispaclitaxel or vincristine.
 5. The method of claim 1 wherein theproteasome inhibitor is bortezomib.
 6. The method of claim 1 wherein theprotein kinase inhibitor is staurosporine.
 7. The method of claim 1wherein the competitive agonist of the estrogen receptor is tamoxifen.8. The method of claim 1 wherein PAC-1 and the second active agent actsynergistically upon administration to thereby treat the cancer.
 9. Themethod of claim 1 wherein PAC-1 and the second active agent areadministered sequentially.
 10. The method of claim 1 wherein the canceris breast cancer, colon cancer, head and neck cancer, kidney cancer,liver cancer, lung cancer, lymphoma, melanoma, osteosarcoma, ovariancancer, pancreatic cancer, renal cancer, or testicular cancer.
 11. Themethod of claim 1 wherein the concentration of PAC-1 is about 2 μM toabout 50 μM.
 12. The method of claim 11 wherein the concentration ofPAC-1 is about 5 μM to about 30 μM.
 13. The method of claim 12 whereinthe concentration of PAC-1 is about 5 μM, about 10 μM, about 12.5 μM,about 25 μM, or about 30 μM.
 14. The method of claim 11 wherein theconcentration of the compound of second active agent is about 1 μM toabout 750 μM.
 15. The method of claim 1 wherein the second active agentand PAC-1 are administered by infusion, by injection, by oraladministration, or a combination thereof.